Hsp90 proteins are the central members of a multi-protein complex called the Hsp90 chaperosome. Several of these Hsp90-client protein complexes are targets for cancer therapies or represent potential targets for future research. The steroid hormone receptors are the best-characterized client proteins of the Hsp90 chaperosome. Drugs designed to target a particular Hsp90 complex or its client protein (oncogenes/kinases/receptors) often produce toxic effects due to non-specific actions on other client protein-Hsp90 complexes. Consequently, a system to assess this "collateral damage" based on drug interactions with Hsp90 and/or its client proteins would be valuable in initial screens designed to eliminate toxic compounds. As a hypothetical example, if a compound (such as a geldanamycin derivative) designed to affect ErbB2-Hsp90 complexes also affected glucocorticoid receptor-Hsp90 complexes with similar potency, the compound would be flagged for potential toxicity and possibly eliminated from further testing. We are developing a "humanized" yeast-based system to assess drugs that may act on the Hsp90 chaperosome and its client proteins. The yeast chaperone components functioning in this pathway are being replaced by their human counterparts to create a relevant model for toxicity studies. Our previous studies with human aryl hydrocarbon receptor, Hsp90 isoproteins, and co-chaperones in yeast provide "proof of concept" for this model toxicity system. The proposed new yeast strains will provide novel, inexpensive, high-throughput toxicity screens that are relevant for the prediction of human toxicity. The Specific Aims of this proposal are 1) to construct yeast strains with human homodimeric steroid hormone receptors that coexpress critical human Hsp90 proteins and co-chaperones and 2) to validate signaling responses in these strains using known Hsp90 inhibitors and steroid hormone agonists and antagonists.